UNIVERSITY    OF   CALIFORNIA    PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  4,  No.  14,  pp.  413-444,  12  figures  in  text  January  24,  1924 


INFLUENCE    OF    REACTION    ON   INTER-RELA- 
TIONS BETWEEN  THE  PLANT  AND 
ITS   CULTURE   MEDIUM 

BY 

•       J.   J.   THERON 

(tontribution  from  the  Laboratory  of  Plant  Nutrition,  College  of  Agriculture) 


I.  INFLUENCE  OF  THE  REACTION  OF  THE  MEDIUM 
UPON  THE  PLANT 

Introduction* 

The  reaction  of  the  substrate  in  which  the  roots  of  plants  develop 
is  of  obvious  importance  to  the  life  of  the  plants.  Earlier  plant  physi- 
ologists have  neglected  this  factor,  and  it  was  not  until  recently  as  a 
result  of  studies  on  the  intensity  of  the  acidity  of  the  soil  solution  of 
certain  soils,  on  the  one  hand,  and  the  relative  resistance  of  different 
varieties  of  plants  to  alkaline  conditions  in  certain  types  of  'alkali 
soils,'  on  the  other,  that  the  significance  of  this  factor  was  fully 
realized. 

Since  the  preliminary  studies  of  Pantanelli26  and  Hoagland,ir'  sev- 
eral investigators  have  attacked  the  problem.  The  practical,  as  well  as 
theoretical  importance  of  a  more  thorough  understanding  of  the  influ- 
ence of  the  reaction  of  the  culture  medium  on  the  growth  and  meta- 
bolism of  plants  seemed  to  warrant  the  investigation  here  described. 

The  object  was  twofold:  (1)  a  study  of  the  effect  of  various  con- 
centrations of  hydrogen  ions  on  the  external  appearance  and  growth 
of  the  more  common  agricultural  plants;  (2)  the  effect  of  the  reaction 
on  the  metabolism  of  these  plants. 


*  The   writer   wishes   to   acknowledge   his   indebtedness   to   Professor   D.    E. 
Hoagland  for  advice  and  kindly  suggestions  during  the  course  of  the  investigation. 


414 


/  niversity  of  California  Publications  in  Agricultural  Sciences       [Vol.4 


For  obvious  reasons,  it  was  impossible  to  employ  more  than  a  few 
types  of  plants  to  accomplish  these  aims;  hence  plants  were  selected 
which  were  adapted  to  the  methods  of  experimentation,  and  which 
may  be  considered  as  representative  of  the  majority  of  field  crops. 
These  were  alfalfa.  (Medicago  sativa),  cotton  (Gossypium  hcrbaccum, 
Durango  variety),  cucumbers  (Cucumus  sativa.  White  Spine  variety), 
Bermuda  grass  (Cyanodon  dactylon),  corn  (Zea  mais.  White  Dent 
field  corn),  barley  {Hordcam  vvlgare,  Beldi  variety),  and  peas  (Pisum 
sativum,  Canada  field),  the  latter  two  being  the  principal  ones  used 
in  the  study  of  the  inter-relations  between  the  metabolism  of  the  plant 
and  the  reaction  of  the  culture  solution. 

Owing  to  the  complexity  of  the  soil  and  the  reactions  taking  place 
therein  and  because  of  the  many  complicating  factors  which  enter  when 
sand  cultures  are  used,  solution  cultures  were  employed  exclusively. 


Experimental 
Baker's  analyzed  salts  and  the  ordinary  distilled  water  of  the 
laboratory  were  used  in  making  all  culture  and  stock  solutions.  The 
stock  solutions  were  those  used  regularly  in  this  laboratory.  Table  1 
gives  the  weights  of  salts  added  to  18  liters  of  water  to  make  up  those 
solutions. 

TABLE   1 

Weights  of  Salts  Dissolved  in  18  Liters  of  the  Distilled  Water  to 

Make  up  the  Stock  Solutions 


Solution  I 


Solution  II 


Solution  III 


KN03 

MgSO, 


1200  grams 
679  grams 


Ca(N03)8    :    1805  grams 


KH,P()4    :    900  grams 


In  table  2  is  given  the  composition  of  the  culture  solution  used 
throughout  in  this  investigation  (except  where  otherwise  stated). 
This  solution  was  made  by  adding  80  c.c.  of  solution  I,  40  c.c.  of  solu- 
tion II,  480  c.c.  of  solution  III.  and  24  grams  of  XaNO..  to  44  liters 

of  water. 

TABLE  2 

Composition  of  Culture  Solution  Expressed  as  Equivalents,  per  Liter 


K 

no3 

H;PO( 

Ca 

Mg 

so, 

Na 

Pn 

.0052 

.0087 

0040 

.0011 

.0007 

0007 

.0064 

4  9 

Iron  was  supplied  in  the  form  of  ferric  tartrate,  one  cubic  centi- 
meter of  a  0.5  per  cent  solution  being  used  per  liter  of  culture  solution. 


1924]      Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        415 

Tn  figure  1,  the  titration  curve  of  the  solution  is  reproduced.  This 
was  obtained  colorimetrieally.  By  interpolation  the  amounts  of  acid 
or  alkali  to  be  added  to  eleven  liters  of  the  solution  to  obtain  any 
desired  PH  within  the  useful  range  can  be  found  from  this  graph. 


45 
40 
35 
30 
25 


Ctty 

h  OH" 


20- 


15 


1 1 L 


pH  of  Solution 


50 


60 

riar.  i 


7.C 


60 


90 


In  an  effort  to  keep  the  composition  of  the  solution  as  constant  as 
possible  over  the  entire  range  of  reactions  used,  the  concentrations  of 
Ca  and  Mg  were  kept  low,  and  were  regulated  by  the  amount   of 


416 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


calcium  which  will  remain  in  solution  at  PH  8.0.  A  precipitate  usually 
occurred  at  PH  8.5  and  often  at  8.0  after  a  few  days.  A  comparatively 
high  concentration  of  phosphate  was  used,  on  the  other  hand,  in  order 
to  increase  the  buffer  effect  of  the  solution.  Unfortunately,  the  buffer 
effect  varies  over  different  ranges  of  reactions. 

This  serious  defect  may  be  partly  remedied  by  the  addition  of  an 
acid  with  a  dissociation  constant  of  about  4.5  and  a  base  with  a  con- 
stant of  about  5.5.  The  only  non-toxic  acids  having  the  desired  con- 
stant are  organic  acids,  e.g.,  citric  acid.  Owing  to  the  danger  of 
excessive  bacterial  growth  in  solutions  containing  organic  matter, 
however,  these  non-toxic,  organic  acids  cannot  be  used  satisfactorily 
(Salter  and  Mcllvaine27).  Ammonium  hydroxide  may  be  used  to 
supplement  the  buffer  effect  of  the  phosphate  at  PH  8  to  PH  10,  but  the 
advantages  to  be  gained  here  are  small  and  the  presence  of  the  ammo- 
nium ion  may  introduce  complicating  factors. 

Growth  in  the  culture  solution  was  very  satisfactory  if  changes 
were  made  weekly.  Sulfuric  acid  and  sodium  hydroxide  were  used 
to  regulate  the  PH  values  of  the  solutions.  Measurements  of  the  reac- 
tion were  made  by  the  indicator  method  of  Clark  and  Lubs.13  Frequent 
use  was  also  made  of  a  Ilildebrand-type  hydrogen  electrode. 

The  plants  were  germinated  between  sheets  of  wet  paper  toweling 
and  the  usual  methods  of  solution  culture  technique  followed.  At 
first  properly  covered  Mason  jars  of  950  c.c.  capacity  were  used  as 
containers.  In  each  jar,  three  plants  were  grown,  ten  jars  being 
employed  for  each  PH  tested. 

The  plants  were  grown  in  series  of  solutions  having  the  following 
initial  values:  4.0,  4.5,  5.0,  6.0,  7.0,  8.0,  8.5,  and  9.0.  Since  the  reaction 
changed  very  rapidly  in  the  direction  of  neutrality,  the  solutions  were 
renewed  every  second  day.  These  frequent  renewals,  however,  did 
not  prevent  the  reactions  of  the  solutions  from  being  changed  consid- 
erably during  the  later  stages  of  growth.  The  maximum  changes  in 
reactions  are  tabulated  in  table  3.  The  plants  were  grown  from  3  to  4 
weeks,  within  which  time  sufficient  growth  was  made  to  determine  ;it 
which  reactions  they  were  affected  adversely. 


TABLE  3 

Maximum  and  Minimum  Values  of  the  Reactions  at  Time  of  Change 


Initial  Ph  of  Series 

4.0 

4.5 

5.0 

6.0 

7.0 

S.O 

8.5 

Maximum  and  minimum 
Ph  at  time  of  change 

4.1-4.4 

4.6-5.0 

5.0-5.2 

6.0-6.1 

7.0-6.9 

8.0-7.9 

8.4-8.0 

1024]     Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        417 

We  assume  naturally  that  the  slightly  increased  concentrations  of 
Na  ions  and  S04  ions  used  in  regulating  the  PH  values  of  the  different 
solutions  have  no  effect  on  the  plant,  and  that  all  differences  in  the 
external  characteristics  are  caused  directly  or  indirectly  by  the  activi- 
ties of  the  hydrogen  or  hydroxyl  ions.  The  influence  of  the  different 
reactions  was  determined  by  the  relative  weights  of  plants  grown  in 
the  different  solutions,  the  length  and  appearance  of  the  roots,  and 
the  height  and  color  of  the  tops. 

The  general  effect  of  excessive  acidity  is  very  characteristic,  and 
is  the  same  for  all  the  plants  used  in  the  experiment.  If  the  culture 
solution  is  injuriously  acid,  the  roots  thicken  and  soon  become  a  dull 
white  in  color  which  is  easily  distinguishable  from  the  silky  white 
appearance  of  normal  roots.  Depending  upon  the  degree  of  acidity, 
the  roots  may  stop  growing  in  length  entirely  or  may  grow  only  sloAvly. 
In  the  latter  case,  they  become  knobby,  because  of  the  excessive 
development  of  laterals  which  penetrate  the  outer  layers  of  the  root 
with  apparent  difficulty.  Lateral  roots  may  develop  to  within  a  few 
millimeters  from  the  growing  tip.  If  the  injury  is  not  too  severe  the 
roots  recover  very  rapidly  when  placed  in  a  more  favorable  solution. 
The  tops  of  the  plants  show  a  marked  stimulation  in  growth  and 
general  vigor,  as  a  rule,  when  compared  with  the  plants  grown  in  a 
more  favorable  solution.  The  stimulation,  however,  is  of  short  dura- 
tion and  after  two  weeks  they  begin  to  lag  behind.  Similar  results 
were  obtained  by  Hixon.14 

An  injurious  alkalinity  of  the  culture  solution  is  very  readily 
recognized  by  a  yellowish  discoloration  of  the  roots.  In  extreme  cases, 
the  roots  become  gelatinous  and  soon  disintegrate.  At  first  the  tops 
showed  no  differences  in  size  and  vigor  as  a  result  of  injury  to  the 
roots  when  compared  with  the  tops  of  plants  growing  in  a  more 
favorable  solution.  After  two  to  three  Aveeks,  however,  a  decided 
stunting  was  noticeable,  and  chlorosis  of  the  new  leaves  set  in. 

Chlorosis  is  generally  ascribed  to  the  lack  of  available  iron.  This 
was  probably  the  main  cause  of  the  chlorosis  of  those  plants  grown  in 
the  alkaline  series.  That  excessive  concentrations  of  hydroxyl  ions, 
however,  may  cause  chlorosis  directly  seems  certain  from  the  following 
considerations. 

A  distinct  test  for  iron  could  be  demonstrated  in  a  solution  kept 
at  PH  8.5  even  after  chlorotic  plants  had  been  growing  in  it  for  a 
week.  Cucumbers  and  alfalfa  will  show  chlorosis  at  PH  7  within  two 
weeks;  at  this  reaction  neither  barley  nor  peas  show  any  chlorosis 


418  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

even  after  nearly  two  months'  growth.  By  this  time,  one  would  expect 
the  supply  of  iron  stored  in  the  seeds  of  the  latter  plants  to  be 
depleted.  Gile  and  Carrero8  found  that  ferric  tartrate  supplied  the 
necessary  iron  to  plants  grown  in  solutions  which  they  thought  to  be 
alkaline. 


-i r 


"i r 


1 r 


100 


60 


40 


zo 


G-reenWts 

of 

Tops 

in  &rs- 


ph  of  Solution. 


30 


40 


30 


60 

Fig.  2 


7.0 


5.0 


10 


It  may  be  objected  that  the  iron  is  not  translocated  from  the  roots 
to  the  tops  in  the  case  of  the  plants  growing  in  an  alkaline  solution, 
and  hence  the  plants  are  nevertheless  suffering  from  a  lack  of  iron. 
The  reaction  of  the  root  juices,  expressed  after  freezing,   indicates. 


19L'4]     Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Mediuvi        419 

however,  that  it  is  hardly  possible  for  the  increased  concentration  of 
the  hydroxy!  ions  to  interfere  with  the  translocation. 

All  the  plants  grown  on  the  acid  side  of  PH  6.0  were  deep  green; 
above  this  reaction  the  color  gradually  became  paler  green,  merging 
into  complete  chlorosis  at  the  higher  PH  values. 

It  is  apparent  that  the  plant  is  influenced  strongly  by  the  reserve 
store  of  food  material  in  the  seed.  Great  care  must  be  taken  in  making 
any  deductions  from  the  experiments  in  which  the  plants  have  been 
grown  for  a  short  period  of  time  only. 

A  much  more  thorough  study  of  the  problem  lias  been  made  using 
the  technique  described  below.  The  results  of  the  experiments  just 
discussed  are  therefore  summarized  in  table  4  without  further  detail 
here. 

TABLE  4 
Effect  of  Acidity  and  Alkalinity  on  Growth  of  Various  Plants 


Plant 


Alfalfa 
Cotton 
Cucumbers 
Barley 
Bermuda  grass 


Ph 
injuriously 

acid 


4.2-4.5 
4.2-4.5 
4  2-4  5 
4.2-4  5 
4.2 


Ph  at  which 

optimum 

growth 

takes  place 


4.8-6.0 
5  0-7  0 
4.8-6  0 
4.5-7.0 
4.5-8.0 


Ph 

injuriously 
alkaline 


7  0 

S  I) 

7  0 

S  0 

'.)  o 


Remarks 


Very  sensitive 
Fairly  resistant 
\  i  n  sensitive 
Resistant 
Highly  resistant 


All  the  varieties  of  plants  tested,  except  the  Bermuda  grass,  were 
affected  adversely  by  approximately  the  same  intensity  of  acidity. 
Alfalfa  and  cucumbers  were  affected  much  more  severely,  a  fact  which 
is  correlated  with  their  greater  sensitiveness  to  alkaline  conditions. 
In  all  cases,  the  best  growth  was  made  when  the  reaction  of  the  culture 
solution  was  between  PH  5  and  PH  6.  It  may  be  of  interest  here  to 
note  that  Fred  and  Davenport'1  found  the  critical  point  for  the  growth 
of  alfalfa  bacteria  to  be  at  PH  4.9.  This  reaction  is  well  within  any 
possible  critical  range  for  the  host  plant. 

The  use  of  the  technique  described  above  involves  an  excessive 
amount  of  labor  and  errors  are  unavoidable.  At  best,  we  are  unable 
to  control  the  reactions  of  the  solutions  satisfactorily.  The  advantages 
of  the  technique  evolved  later  and  described  below  will  at  once  be 
evident. 

Whereas  with  the  former  technique,  30  plants  were  grown  in  ten 
different  jars  at  every  PH  value  in  the  experiment,  all  30  plants  were 
now  grown  in  one  three-gallon  (eleven  liter)  earthenware  crock.     The 


420  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

plants  were  supported  as  before  in  perforations  on  a  cork  sheet  made 
by  binding  together  three  12"  x  4"  x  %"  cork  slabs  with  two  strips 
of  wood  nailed  on  the  edges.  To  prevent  lateral  movements,  small 
pieces  of  wood  were  nailed  on  the  underside  of  the  overlapping 
corners.  Slabs  of  wood  %"  thick  serve  the  purpose  even  better,  since 
the  plants  can  be  supported  more  firmly  in  them.  These  slabs  must  be 
soaked  thoroughly  in  hot  pure  paraffin  so  as  to  prevent  the  absorption 
of  water.  By  growing  all  thirty  plants  in  this  large  volume  of  solu- 
tion, the  effect  of  the  inherent  variability  of  the  plants  is  minimized 
and  most  of  the  experimental  errors  are  eliminated.     The  roots  can 


40 

30k' 
Z0 


w 


Lengths 

Rgf0T5  BARLtY    -Grown  44  Days 

in  Cm  CORN       •    -     2.6    ■ 


PEAS      -    •     27    ■ 

dH  of  Solution 


40  30  60  7.0  SO  90 

Fig.   3 

be  inspected  readily  and  the  PH  of  the  solution  can  be  adjusted  con- 
veniently, rapidly,  and  as  frequently  as  desired.  The  PH  is  adjusted 
by  withdrawing  5  c.c.  samples  of  the  solution  and  determining  the 
reaction  colorimetrically.  The  amounts  of  acid  or  alkali  which  must 
be  added  to  bring  the  PH  to  the  original  value  are  read  off  from 
figure  1,  and  the  required  quantities  added  to  the  solution.  Figure  1 
applies  strictly  to  only  the  fresh  solution.  Within  a  week,  however,  the 
composition  of  the  solution  did  not  change  sufficiently  to  invalidate 
the  method.  The  solutions  were  changed  every  week  and  the  PH 
adjusted  twice  a  day,  i.e.,  in  the  morning  and  evening.  During  the 
later  stages  of  growth,  this  becomes  necessary  more  frequently  in  the 
case  of  plants  growing  at  the  reactions  PH  4.0-5.0  and  PH  S. 0-9.0. 
Over  these  ranges,  the  buffer  effect  of  the  solution  is  relatively  small 
and  the  power  of  the  plant  to  change  the  PH  of  the  solution  is  increased 
iscc  figs.  4  and  5).  All  the  plants  experimented  with  showed  a  tend- 
ency to  change  the  PH  of  the  solutions  to  a  value  between  PH  (>.'_' 
and  6.8.  ' 


1924]     Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        421 

Because  of  the  lack  of  time,  it  was  not  possible  to  subject  all  the 
plants  used  in  the  former  experiments  to  these  better  controlled 
methods.  This  was  done,  however,  with  four  widely  different  types 
of  plants,  namely,  barley,  peas,  cucumbers,  and  corn. 


60 


60 


40 


20 


Cc.ty 


* 

pM  of  Solution 
■ i 


60 


40 


ZD 


30 


40 


30  £0 

Fig.  4 


9.0 


In  table  5,  one  experiment  with  peas  is  summarized.  In  the  first 
column  are  given  the  desired  reactions  of  the  solution,  in  the  second 
the  highest  and  lowest  PH  values  reached  during  the  course  of  the 
experiment,  and  in  the  seventh  the  number  of  cubic  centimeters  of 
normal  hydrogen  (sulphuric  acid),  or  normal  hydroxide  ions  (sodium 
hydroxide),  added  during  the  entire  period  of  growth  to  replace  that 
neutralized  by  the  plants. 


422 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


In  figure  2,  the  green  weights  of  the  tops  of  30  plants  of  the  four 
types  are  plotted  against  the  PH  of  each  series  (see  column  1,  table  5), 
and  in  figure  3,  the  length  of  the  roots.  Since  neither  the  change  of 
PH  nor  the  change  in  the  total  molality  of  acid  or  base  with  time  is  an 
arithmetic  function,  it  is  impossible  to  calculate  an  average  PH.  The 
true  average  PH  values  differ  only  by  a  small  amount  from  the  desired 
PH  such  that  the  given  curves  are  not  greatly  different  from  the 
curves  which  would  be  obtained  if  the  true  average  reactions  were 
used.    The  differences  are  within  the  limits  of  the  experimental  error. 


TABLE  5 

Summary  of  a  Typical  Experiment  with  Peas 


Desired 
Pnof 

Maxi- 
mum 
range 
of  Ph 

Days 
grown 

No.  of 
plants 

Green 
weight 
of  tops 
gms. 

Length 

of 

roots 

cms. 

C.C.  N/1 

Reagent 

neutralized 

Remarks 

series 

Acid 

Alkali 

3.9 

3.9-4  0 

25 

30 

82.8 

28 

14.8 

Roots  severely  injured 

4.5 

4.5-4.7 

25 

30 

86.2 

35 

8.8 

Very   slight    injury    to 
roots 

5.0 

5.0-5.2 

25 

30 

95.3 

40 

8.9 

Best  growth 

6.0 

6  0  6  1 

25 

30 

94.2 

40 

7.0 

Best  growth 

7.0 

7.0-6.9 

25 

30 

81.1 

38 

8.0 

8.0 

8.0-7.9 

25 

30 

59.4 

25 

60.0 

Tops  slightly  chlorotie 

8.5 

8.5-8.3 

25 

30 

27.6 

20 

41.0 

Roots  badly  injured. 
Tops  chlorotie 

The  juices  of  the  plants  were  needed  for  other  experiments,  so 
the  dry  weights  were  not  determined.  For  the  present  purpose,  the 
green  weights  of  the  tops  give  a  reliable  criterion  of  the  general  vigor 
and  size  of  the  plants.  The  differences  in  the  weights  of  the  barley 
and  pea  plants  can  hardly  be  considered  as  significant  in  themselves 
on  account  of  the  inherent  variability  of  the  plants.  If  the  observa- 
tions on  the  other  effects  are  taken  into  consideration,  however,  it 
becomes  evident  that  the  small  differences  in  weight  are  true  expres- 
sions of  the  effect  of  the  corresponding  reactions  on  the  growth  of 
the  plants. 

The  maximum  changes  in  Ph  brought  about  by  all  four  types  were 
the  same  as  that  given  for  peas  in  column  2,  table  ."),  excepl  in  the 
case  of  corn,  grown  in  the  alkaline  solution,  where  the  reaction  fre- 
quently reached  the  PH  8.1.  Since  tour  widely  different  types  of 
plants  were  used,  the  curves  may  be  considered  as  a  definite  measure 
of  the  effect  of  the  reaction  of  the  culture  medium  on  the  growth  of 


1924]      Therein:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        423 

most  agricultural  plants  as  indicated  by  the  yield.  They  show  unmis- 
takably that  the  optimum  range  of  the  reaction  for  the  propagation 
of  these  plants  in  solution  cultures  is  between  PH  4.5  and  6.0,  and 
agree  substantially  with  the  results  found  with  the  earlier  method  of 
experimentation  (see  table  4).  In  figure  4,  the  amounts  of  normal 
acid  or  alkali  neutralized  by  30  plants  during  the  first  25  days  of 


Fig.  5 


growth  is  represented  graphically  for  each  type.  These  curves  are 
not  strictly  comparable,  since  the  plants  were  grown  at  different  times 
of  the  year.  The  amounts  of  acid  or  alkali  neutralized  within  a 
definite  period  of  time  depend  largely  upon  the  rapidity  of  growth. 

Although  the  curves  are  onry  of  a  qualitative  significance,  they 
are  very  expressive  of  the  power  of  the  plant  to  overcome  any  unfavor- 
able acidity  or  alkalinity,  especially  the  latter.  This  power  is  of 
obvious  importance  to  the  plant  and  must  form  an  integral  part  of 
any  study  of  acid  or  alkali  resistant  crops,  either  in  the  soil  or  in 


424  University  of  California  Publications  in  Agricultural  Sciences       [Yo\.i 

solution  culture.  Under  natural  conditions,  the  plant  has  to  contend 
with  the  reaction  of  the  medium  in  which  its  roots  are  immersed  or 
imbedded  from  the  time  of  germination  to  maturity.  If  the  medium 
is  sufficiently  highly  buffered  or  is  continually  renewed,  such  that 
little  or  no  change  of  reaction  is  brought  about  under  the  influence 


pHof 
Sap 


CO 


50 


BARLCY 


M 


pH  of  Solution 


40 


50  60 

Fig-.  6 


7.0 


flO 


Fie. 


of  the  plant,  the  ability  to  overcome  any  unfavorable  reaction  is  cor- 
rectly expressed  by  these  curves.  From  a  purely  theoretical  point  of 
view,  however,  this  ability  may  be  determined  at  different  reactions 
for  plants  treated  similarly  up  to  the  time  of  experimentation,  so 
that  the  vigor  and  internal  mechanism  of  all  the  plants  will  as  nearly 


]<»24|      Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        42") 

as  possible  be  the  same  when  subjected  to  the  different  acidities  or 
alkalinities.  These  must  be  such  that  the  plant  mechanism  will  not 
be  injured  or  altered  materially  during:  the  period  of  experimentation. 

Five  sets  of  25  barley  plants .  each  were  grown  in  earthenware 
crocks  of  71/-)  liter  capacity.  All  the  solutions  had  a  reaction  of 
PH  6.8,  and  were  changed  weekly.  When  plants  were  four  weeks 
advanced,  the  sets  were  transferred  to  solutions  having  the  reactions 
4.0,  5.0,  6.0,  7.0,  and  8.0,  and  these  were  kept  as  constant  as  possible 
for  four  days  by  the  addition  of  N/5  acid  or  alkali.  It  was  assumed 
that  the  sets  of  plants  were  not  affected  materially  by  the  differences 
in  reactions  within  this  period  of  time. 

In  figure  5,  the  amounts  of  N/5  acid  or  alkali  neutralized  are 
plotted  against  the  desired  PH  as  before.  Unfortunately  the  number 
of  determinations  made  are  insufficient  to  permit  of  the  smoothing 
out  of  the  curves.  Their  general  shape,  however,  is  obvious.  On  the 
alkaline  side  of  PH  6.8,  the  ability  to  neutralize  excessive  concentra- 
tions of  hydroxyl  ions  increases  very  rapidly  with  the  increase  in  PH 
and  probably  does  not  reach  a  maximum  even  at  PH  8.0.  On  the 
acid  side,  however,  the  increase  is  less  rapid  and  reaches  a  maximum 
between  PH  4.0  and  5.0. 

Influence  of  Factors  other  than  the  Reaction 

The  plants  were  grown  in  the  open  during  the  summer  months 
and  in  a  heated  greenhouse  during  winter.  In  the  course  of  the 
investigation,  it  became  evident  that  plants  grown  at  different  seasons 
show  slight  differences  in  their  resistance  to  the  effect  of  the  reaction. 
This  is  most  probably  due  to  the  differences  in  the  rate  of  growth 
under  different  atmospheric  conditions. 

The  influence  of  the  composition  of  the  culture  solution  on  the 
effect  of  the  reaction  was  not  determined,  as  only  one  solution  was 
used  throughout  the  investigation.  It  is  highly  improbable,  however, 
that  the  composition  of  the  solution,  within  wide  limits,  is  a  factor 
in  any  of  the  divers  phases  of  this  study.  The  results  obtained  by 
Salter  and  Mellvaine27  and  those  obtained  by  the  writer  seem  to 
substantiate  this  assumption. 

The  amounts  of  water  transpired  by  plants  from  solutions  of 
different  reactions  were  found  to  be  the  same  within  the  limits  of  the 
experimental  error. 


426  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


Discussion 

The  conclusion  reached  by  earlier  workers2' 4-  22  was  that  the  H 
ion  was  more  toxic  than  the  OH  ion  to  plants  growing  in  solution 
cultures.  Their  results,  however,  are  untenable  because  they  failed 
to  distinguish  between  potential  and  actual  acidity  or  alkalinity.  The 
ability  of  the  plant  to  change  the  reaction  of  the  nutrient  medium 
was  likewise  overlooked. 

In  a  series  of  papers,  Hoagland15,  16, 17>  1S  has  called  attention  to 
both  these  factors  and  showed  that  the  OH  ion  is  much  more  toxic 
to  barley  seedlings  in  solution  culture  than  the  H  ion.  An  OH 
ion  concentration  greater  than  PH  8.2  was  distinctly  injurious, 
whereas  an  H  ion  concentration  of  Pn  5.0  was  found  to  be  favorable 
to  growth  and  to  cause  no  injury.  Similar  results  were  obtained  by 
Duggar5  using  various  types  of  solutions  and  growing  the  plants 
under  the  most  diverse  environmental  conditions.  One  of  the  most 
complete  and  satisfactory  studies  on  this  problem  is  that  of  Salter  and 
Mcllvaine.27  These  investigators  experimented  with  corn,  wheat, 
soybeans,  and  alfalfa,  growing  the  plants  at  seven  different  II  ion 
concentrations.  The  plants  were  grown  for  relatively  short  periods  of 
time  and  the  solution  changed  once  every  four  days.  A  distinct  maxi- 
mum in  the  growth  of  the  plants  was  found  at  PH  5-PH  6.  At  a  neutral 
reaction,    decided    decreases    in    the    yields    could    be    demonstrated. 

We  have  already  called  attention  to  the  advisability  of  growin-a- 
the  plants  for  a  considerable  length  of  time  so  as  to  overcome  the 
influence  of  the  food  supply  stored  in  the  seed.  Only  in  this  way 
is  it  possible  to  obtain  a  true  measure  of  the  effect  of  the  reaction  of 
the  solution.  The  growth  periods  employed  by  these  investigators 
were  undoubtedly  too  short.  On  the  other  hand,  the  variations  in 
reaction  caused  by  young  plants  are  relatively  small,  so  that  plants 
grown  in  accordance  with  the  technique  they  employed  will  give  more 
reliable  results  if  the  experiment  is  discontinued  after  two  weeks 
than  if  the  plants  are  grown  for  a  longer  period  of  time.  Our  results 
agree  substantially  with  those  of  these  investigators. 

Hixon14  found  a  distinct  minimum  in  the  development  of  young 
plants  as  measured  by  the  growth  in  length  of  the  roots  and  tops  at 
PH  5  for  Pisum  and  PH  6  for  most  other  plants.  This  minimum  point 
is  interpreted  as  that  of  greatest  efficiency  and  the  point  of  normal 
growth.  We  have  been  able  to  confirm  his  results  in  part.  A  decided 
stimulation  occurred  at  acidities  which  injured  the  plants  definitely 


1924]     Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        427 

later  on.  No  stimulation  was  noticed  in  the  tops  of  plants  grown  in 
alkaline  solutions.  The  roots  were  occasionally  longer  than  those 
of  the  plants  grown  at  PH  5  or  PH  6. 

A  glance  at  figures  4  and  5  is  .sufficient  to  make  evident  the  import- 
ance of  controlling  the  reaction  of  the  solution  under  investigation. 


Fig.  8 


428  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

Changes  of  solution  every  fourth  or  fifth  day  are  obviously  insufficient 
to  maintain  the  PH  constant  even  approximately,  when  the  plants 
are  three  to  four  weeks  advanced.  With  small  volumes  of  solutions 
supporting  relatively  large  numbers  of  plants  grown  at  PH  5,  this 
becomes  increasingly  difficult.  In  an  investigation  by  McCall  and 
Haag,23  this  point  seems  to  be  lost  sight  of  completely.  From  their 
investigations,  it  appears  that  wheat  plants  grow  best  at  reactions 
between  PH  3  and  PH  4.  It  is  very  plain,  however,  that  the  reactions 
of  the  solutions  in  the  neighborhood  of  the  roots  must  have  been  very 
different  from  what  they  were  assumed  to  be.  It  is  not  strange  that 
the  solution  with  the  highest  buffer  effect  gave  the  poorest  growth. 

In  culture  solutions,  the  diffusion  of  solutes  is  relatively  rapid 
and  as  a  rule  the  reaction  around  the  roots  is  the  same  as  that  in  the 
bulk  of  the  solution.  If,  however,  the  free  diffusion  is  interfered 
with,  such  as  often  happens  among  the  roots  in  the  upper  few  inches 
of  the  solution,  the  reaction  may  be  very  different  in  this  region  from 
what  it  is  in  the  bulk  of  the  solution.  Over  the  ranges  of  low  buffer 
effect,  a  difference  of  0.5  PH  can  occasionally  be  demonstrated  under 
such  conditions.  In  soils,  the  diffusion  is  infinitely  slower  and  the 
reaction  of  the  solution  in  contact  with  the  absorbing  roots  will  be 
determined  solely  by  the  ability  of  the  plant  to  overcome  the  buffer 
effect  of  the  soil  complex  in  its  immediate  vicinity.  Considering  the 
power  of  growing  plants  to  regulate  the  Ph  value  of  the  culture 
medium,  the  conclusion  is  inevitable  that  the  direct  effect  of  the 
actual  reaction  of  most  soils  can  hardly  be  a  factor  in  the  complex 
which  determines  the  growth  of  the  plant  in  that  soil,  provided  the 
plant  has  the.  ability  to  establish  itself  firmly.  In  this  connection  the 
work  of  Joffe10  with  alfalfa  is  very  elucidating.  The  results  obtained 
with  solution  cultures  agree  well  with  those  of  this  investigator  using 
soils  acidified  artificially. 

From  the  determination  of  the  reactions  of  numerous  acid  soils 
reported  by  Gillespie,"  and  Sharp  and  Hoagland,28  it  is  apparent 
that  the  reaction  of  the  majority  of  these  soils  can  have  little  or  no 
direct  effect  on  the  growth  of  plants.  The  infertility  of  acid  soils 
can  usually  be  ascribed  to  causes  other  than  the  H  ion  concentra- 
tion. The  solubility  of  aluminum  in  the  slightly  acid  soil  solutions 
of  these  soils  is  undoubtedly  responsible  for  some  of  the  phenomena 
attributed  formerly  to  the  acidity  of  the  soil.1'  12,  "■  25,  29 

The  soil  solution  of  many  alkali  soils  has  a  highly  alkaline  reaction, 
which  tends  to  prevent  the  young  plants  from  germinating  or  develop- 


1924]      Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        429 

ing.  Germinating  seeds  have  a  remarkable  ability  to  change  the 
reaction  of  the  alkaline  medium  in  which  they  are  immersed,  in  the 
direction  of  neutrality,  so  that  the  PH  value  around  the  seeds  may 
be  made  favorable  to  germination.     The  ability  of  the  seedling  to 


Fig.  9 


regulate  the  reaction  is  comparatively  small  and  hence  the  young 
roots  may  be  unable  to  penetrate  beyond  the  regions  of  the  favorable 
reaction  brought  about  by  the  seed.  If  the  soil  solution  has  both  a 
high  PH  value  and  a  high  concentration  of  salts,  the  seedlings  Avill 
natnrallv  be  unable  to  survive. 


430  University  of  California  Publications  in  Agricultural  Scioices        [Vol.4 

Effect  of  Reaction  of  Culture  Solution  on  the  Reaction  and 
Buffer  Effect  of  the  Plant  Juices 

The  plants  from  the  experiments  described  above  were  frozen 
immediately  after  they  were  harvested.  This  was  done  in  a  cold  room 
kept  at  12°  F.,  from  which  they  were  only  removed  as  they  were 
needed.  The  plant  juices  were  obtained  by  grinding  the  frozen  mass, 
thawing  this  rapidly  in  a  warm  room,  and  then  expressing  the  sap  by 
hand  through  a  few  thicknesses  of  cheesecloth.  All  determinations 
were  made  as  soon  as  possible  after  the  frozen  ground  material  was 
thawed  out. 

THE  H-ION  CONCENTRATION  OF  THE  SAP 

The  reaction  of  the  juices  of  the  roots  and  tops,  obtained  in  the 
above  way,  was  measured  by  means  of  a  Hildebrand  hydrogen  elec- 
trode. Difficulty  was  experienced  in  making  the  measurements  as 
reduction  of  N03  ions  apparently  took  place  on  the  electrode.  This 
was  especially  true  in  the  case  of  the  juices  from  those  plants  grown 
at  the  acid  reactions.  This  difficulty  was  obviated  to  some  extent  by 
leaving  the  NaNO:.  out  of  the  culture  solution  during  the  last  week 
of  the  experiments. 

In  figures  6  and  7,  the  reactions  of  the  tops  and  roots  of  cucumber, 
barley,  pea,  and  corn  plants  grown  at  different  reactions  are  repre- 
sented graphically.  The  reactions  of  the  sap  expressed  from  the  tops 
were  not  influenced  by  the  reaction  of  the  culture  solution,  the  varia- 
tions in  reaction  being  within  the  limits  of  the  experimental  error. 

On  the  other  hand,  the  reactions  of  the  root  juices  are  decided ly 
changed  by  the  reaction  of  the  solution.*  It  is  plain,  however,  that 
the  reactions  of  the  roots  are  very  different  from  the  reactions  of  the 
solutions,  except  when  these  are  between  PH  6  and  PH  7.  Whether 
the  reaction  of  the  root  juices  is  influenced  according  to  any  definite 
rule  by  the  reaction  of  the  solution,  as  may  be  suggested  by  the  curve 
for  pea  roots,  it  is  impossible  to  say  at  present,  owing  to  the  relatively 
large  experimental  error  involved  in  these  measurements.  Truog  and 
Meacham,31  after  studying  the  effect  of  additions  of  lime  to  a  soil, 
concluded  that  the  reaction  of  the  soil  can  influence  the  reaction  of  the 
sap  expressed  from  the  tops  of  the  plants.  It  seems  obvious,  however, 
that  the  differences  in  the  reactions  from  the  limed  and  unlimed  plots 


*  Compare   Bryan,   O.   O,   Effect   of  different   reactions   on   the   growth    and 
nodule  formation  of  Soy  beans.     Soil  Science,  vol.  12,  no.  4,  pp.  271-2S7  (1922). 


1924]     Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        431 

are  within  the  experimental  error,  apart  from  the  fact  that  many 
other  factors  enter  in  the  case  of  plants  growing  in  limed  and  un limed 
acid  soils. 


Fig.   10 


432  rniver.siti/  of  California  Publications  in  Agricultural  Sciences        [Vol.4 


THE  BUFFER  EFFECT  OF  THE  SAP 

The  juice  obtained  from  the  plants  in  the  way  described  was 
titrated  electrometrically  (after  an  equal  volume  of  water  had  been 
added)  with  N/20  acid  and  alkali.  The  PH  value  was  invariably 
increased  by  about  one-tenth  of  a  magnitude  of  the  dilution. 


Fig.  11 


In  figures  8  and  9  the  lit  ration  curves  for  barley  tops  and  roots, 
respectively,  are  given.  The  curves  are  represented  as  if  25  c.c.  of 
undiluted  sap  had  been  titrated  with  N/10  reagents.  The  correspond- 
ing curves  for  peas  are  given  iii  figures  10  and  11. 


1924]      Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        433 

Hempel13  has  shown  that  the  buffer  effect  of  plant  juices  is  mainly 
due  to  the  organic  acids  and  salts  of  these  acids  contained  in  the  plant 
system.  It  appears  from  figure  8  that  the  reaction  of  the  nutrient  solu- 
tion has  influenced  the  concentration  of  those  acids  with  dissociation 
constants  less  than  10"  very  markedly  in  the  tops  of  barley  plants 
although  the  reaction  of  the  expressed  sap  is  apparently  unchanged. 


Fig.   12 


In  the  roots  the  buffer  effect  is  also  influenced.  Here,  however,  only 
those  acids  with  a  dissociation  constant  higher  than  10~6  are  affected. 
In  the  case  of  the  pea  plants,  neither  the  reaction  nor  the  buffer  effect 
of  the  sap  expressed  from  the  tops  was  influenced  by  the  reaction  of 
the  nutrient  medium.  The  roots  on  the  other  hand  were  affected 
similarly  to  the  barley  roots.  These  plants  were  grown  in  a  green- 
house during  the  winter. 

In  figure  12,  the  results  of  a  similar  experiment  with  peas  grown 
in  the  open  in  summer  are  given.     In  this  experiment,  the  reaction  of 


434  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

the  sap  expressed  from  the  tops  was  unchanged,  but  the  buffer  effect 
was  influenced  by  the  reaction  of  the  culture  solution.  The  effect, 
however,  is  the  reverse  of  what  it  was  in  the  case  of  the  tops  of  the 
barley  plants,  and  in  both  instances  was  only  noticeable  in  the  con- 
centration of  those  acids  with  a  dissociation  constant  lower  than  10~6. 
Unfortunately,  it  was  not  possible  to  pursue  this  line  of  investiga- 
tion with  additional  plants  and  under  the  different  atmospheric  con- 
ditions. It  seems,  however,  that  a  thorough  study  along  these  lines 
will  throw  considerable  light  on  the  salt  metabolism  of  plants. 

Summary 

1.  The  influence  of  the  reaction  of  the  culture  medium  on  the 
growth  and  metabolism  of  the  common  agricultural  plants  was  stiidied 
by  growing  typical  plants  in  solution  cultures  at  different  reactions. 

2.  After  experimenting  with  several  different  methods,  a  technique 
was  devised  by  which  the  reaction  of  the  solution  could  be  conveniently 
controlled.  Particular  attention  was  given  to  the  constant  mainten- 
ance of  the  desired  hydrogen-ion  concentration  during  the  experi- 
mental periods. 

3.  Plants  grown  in  solution  cultures  have  an  optimum  growth 
reaction  at  PH  4.5  to  PH  6. 

4.  The  reaction  of  the  juice  expressed  from  the  tops  of  the  plants 
was  not  influenced  by  the  reaction  of  the  culture  medium,  whereas 
the  reaction  of  the  juices  expresesd  from  the  roots  was  modified 
considerably. 

5.  The  buffer  effect  of  both  the  roots  and  the  tops  may  be  influ- 
enced by  the  reaction  of  the  culture  solution.  In  the  tops,  the  acid 
reserve  is  affected  and  in  the  roots,  the  alkali  reserve. 

6.  Observations  were  made  on  the  ability  of  the  growing  plant  to 
chanffe  the  reaction  of  either  acid  or  alkaline  culture  solutions. 


1924]      Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        436 


LITERATURE  CITED 

i  Abbott,  J.  B.  Connor,  S.  D.,  and  Smallet,  H.  E. 

1913.  Soil  acidity,  nitrification  and  the  toxicity  of  soluble  salts  of  aluminum. 

Indiana  Agr.  Exp.  Sta.  Bull.  170,  pp.  329-374. 
-  Breazeale,  J.  P.,  and  LeClerc,  J.  A. 

1912.     The  growth  of  wheat  seedlings  as  affected  by  acid  or  alkaline  con- 
ditions.    U.  S.  Dept.  Agr.,  Bur.  Chem.,  Bull.  149. 
s  Clark.  W.  M.,  and  Lubs,  H.A. 

1917.  The  colorimetric  determination  of  hydrogen  ion  concentration  and 

its   application   in  bacteriology.      Jour.   Bact.,   vol.    2,   pp.    1-34, 
109-136,  191-236. 
•»  Dachxowski,  Alfred 

1914.  The  effects  of  acid  and  alkaline  solutions  upon  the  water  relations 

and  the  metabolism  of  plants.     Amer.  Jour.  Bot.   vol.   1,  no.  8, 
pp.  412-440. 
,  =  Duggar,  B.  M. 

1920.     H-ion    concentration    and    the    composition    of   nutrient    solution    in 
relation    to    the    growth    of    seed    plants.      Ann.    Missouri    Bot. 
Garden,  vol.  7,  no.  1,  pp.  1-49. 
e  Fred,  E.  B.,  and  Davenport,  Audry 

1918.  The  influence  of  reaction  on  nitrogen-assimilating  bacteria.      Jour. 

Agr.  Res.,  vol.  14,  no.  8,  pp.  317-336. 
7  Fred,  E.  B.,  and  Loomis,  N.  E. 

1917.  Influence  of  hydrogen  ion  concentration  of  medium  on  the  reproduc- 

tion of  alfalfa  bacteria.     Jour.  Bact.,  vol.  2,  no.  6,  pp.  629-633. 
s  Gile,  P.  L.,  and  Carrero,  J.  O. 

1916.     Assimilation  of  iron  by  rice  from  certain  nutrient  solutions.     Jour. 

Agr.  Res.,  vol.  7,  no.  12,  pp.  503-528. 
9  Gillespie,  L.  J. 

1916.  The  reaction  of  the  soil  and  measurements  of  hydrogen  ion  concen- 

tration.    Jour.  Wash.  Acad.  Sci.,  vol.  6,  no.  1,  pp.  7-16. 
io  Gillespie,  L.  J.,  and  Hurst,  L.  A. 

1918.  Hydrogen-ion   concentration — soil  type — common  potato  scab.     Soil 

Sci.,  vol.  6.  pp.  219-236. 
"  Haas.  A.  R.  C 

1920.     Studies  on  the  reaction  of  plant  juices.     Soil  Sci.,  vol.  9,  no.  5,  pp. 
341-368. 
i-  Hartwell.  B.  L.,  and  Pember,  F.  R. 

1918.     The   presence   of  aluminum    as   a   reason   for   the    difference   in    the 
effect  of  so-called  acid  soil  on  barley  and  rye.     Soil  Sci.,  vol.  6, 
no.  4,  pp.  259-277. 
i  s  Hempel,  Jenny 

1917.  Buffer   processes    in    the    metabolism    of    succulent    plants.      Compt. 

Rend.  Lab.  Carlsberg,  t.  13,  no.  1,  p.  130. 
uHixon,  R.  X. 

1920.     The  effect  of  the  reaction  of  the  nutrient   solution  on  germination 
and   the    first   stages   of  plant   growth.      Med.   K.    Veten.    Nobel 
Inst.,  Band  4,  no.  9,  pp.  1-28. 
is  Hoagland,  T).  R. 

1917.     The  effect  of  the  hydrogen  and  hydroxyl  ion  concentration  on  the 
growth  of  barley  seedlings.     Soil  Sci.,  vol.  3,  no.  6,  pp.  547-560. 


436  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

is  Hoagland,  D.  B. 

1918.  The   relation   of  the  plant   to   the  reaction  of  the   nutrient    solution. 

Science,  n.  s.,  vol.  48,  no.  1243,  pp.  422-425. 

1 "  HOAGLAND,  D.  B. 

1919.  Relation   of  the   nutrient   solution   to  the   composition   and   reaction 

of  cell  sap  of  barley.    Bot.  Gaz.,  vol.  68,  no.  4,  pp.  297-304. 
is  Hoaglaxd,  D.  R. 

1919.  Belation  of  the  concentration  and  reaction  of  the  nutrient  medium 

to   the   growth    and   absorption   of  the   plant.      Jour.    Agr.   Bes., 
vol.  18,  no.  2,  pp.  73-117. 

m  JOPFE,  J.  S. 

192(i.  The  influence  of  the  soil  reaction  on  the  growth  of  alfalfa.  Soil 
Sei.,  vol.  10,  no.  4,  pp.  301-307. 

2°  Jones,  L.  H.,  and  Shive,  J.  W. 

1921.  Effect  of  ammonium  sulphate  upon  plants  in  nutrient  solutions  sup- 
plied with  ferric  phosphate  and  ferrous  sulphate  as  sources  of 
iron.     Jour.  Agr.  Bes.,  vol.  21,  no.  10,  pp.  701-728. 

-i  Kappex,  H. 

1920.  Ueber  die  aziditatsformen  des  Bodens  und  ihre  pflanzenphysiologische 

Bedeutung.     Landw.  Vers.  Stat.,  vol.  96,  pp.  277-307. 
--  Loew,  r.  A. 

1903.  The  toxic  effect  of  H  and  OH  ions  on  seedlings  of  Indian  corn. 
Science,  n.  s.,  vol.  18,  no.  453,  pp.  304-308. 

23  McCall,  A.  G.  and  Haag,  J.  B. 

1921.  The   relation   of   the   hydrogen   ion   concentration   of   nutrient    solu- 

tions to  growth  and  chlorosis  of  wheat  plants.     Soil  Sei.,  vol.  12, 
no.  1,  pp.  69-77. 

24  Mirasol,  J.  J. 

1920.  Aluminum  as  a  factor  in  soil  acidity.  Soil  Sei.,  vol.  10,  no.  3,  pp. 
153-218. 

25  MlYAKE,   K. 

1916.     The  toxie  action  of  the  soluble  aluminum  salts  upon  the  growth  of 
the  rice  plant.     Jour.  Biol.  Chem.,  vol.  25,  no.  1,  pp.  23-28. 
2«  Paxtanelli,  E. 

1915.     Ueber  Ionen  Aufnahme.     Jahrb.  Wiss.  Bot.   (Pringsheini),  Band  5C, 
pp.  689-733. 
2"  Salter,  B.  M.,  and  McIlvaixe,  T.  C. 

1920.     Effect  of  reaction  of  solution  on  germination  of  seeds  ami  growth 
of  seedlings.     Jour.  Agr.  lies.,  vol.  19,  no.  2,  pp.  73-95. 
2s  Sharp,  L.  T.  and  Hoagland,  D.  B. 

.     Acidity  and  absorption  in  soils  as  measured  by  the  hydrogen  elec- 
trode.    Jour.  Agr.   Bes.,  vol.  7,  no.  3,  pp.  123-145. 
2n  Stoklasa,  J. 

1918.     Ueber  den  Einfluss   des  Aluniinnniions  ant'  die   Keimung  des  Samens 
und     ilie    Entwickelung    der    rflanzen.       Biochem.     Ztschl-.,     vol.     B9, 
pp.   137-223. 
SO  Texjog,  E. 

1918.  Soil  Acidity:    I.  Its  relation  to  the  growth  of  plants.     Soil  Sei.,  vol. 

5,  no.  3,  pp.   169-195. 
■"  TRUOG,  E.,   and   MEACHAM,  M.   B. 

1919.  Soil  acidity:     II.    Its  relation  to  the  acidity  of  the  plant  juice.     Soil 

Sei.,  vol.  7,  no.  6,  pp.  469-474. 


1924]      Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        43/ 


II.  POSSIBLE  MECHANISM  OF  THE  PLANT'S  INFLUENCE 
ON  THE  REACTION  OF  THE  CULTURE  SOLUTION 

Recent  studies  on  the  absorption  of  inorganic  ions  by  plants  as 
well  as  the  large  amount  of  work  done  on  the  problem  of  the  antagon- 
ism between  ions  and  the  physiological  balance  in  culture  solutions 
have  thrown  some  light  on  the  mechanism  by  which  the  plant  obtains 
its  inorganic  elements.  The  importance  of  a  more  thorough  knowl- 
edge of  this  process  is  undisputed.  Unfortunately  investigations  on 
this  problem  are  hampered  by  our  meager  knowledge  of  the  true 
nature  of  solutions  and  the  methods  of  analysis  at  our  disposal. 

In  considering  the  absorption  of  any  ion,  account  must  be  taken 
of  the  activities  of  that  ion  inside  and  outside  of  the  membrane 
effective  in  absorption.  We  are  at  present  unable  to  determine  the 
activity  of  any  ion  in  a  system  as  complex  as  a  complete  culture  solu- 
tion except  that  of  the  H  ion,  which  can  usually  be  determined  with 
sufficient  accuracy. 

Since  the  activity  and  the  total  molal  concentration  of  the  H  ion 
are  conveniently  and  rapidly  determined  and  since  we  have  every 
reason  to  believe  that  the  H  and  OH  ions  are  absorbed,  fundamentally, 
in  the  same  way  as  any  other  positive  or  negative  ion,  we  have  here 
a  very  efficient  means  of  studying  this  problem. 

In  Part  I  of  this  investigation,  a  series  of  experiments  were 
described  which  were  concerned  mainly  with  the  effect  of  the  reaction 
of  a  culture  solution  on  the  growth  and  metabolism  of  several  types 
of  plants.  In  the  present  paper,  some  preliminary  experiments  are 
described  which  are  concerned  with  the  effect  of  the  growing  plant 
on  the  composition  and  especially  on  the  reaction  of  culture  solutions. 

Pantanelli8  observed  that  plants  always  changed  the  reaction  of  a 
single  salt  solution  in  the  direction  of  neutrality  except  when 
(NH4)2S04  was  the  solute.  In  a  solution  of  this  salt,  the  reaction 
remained  at  the  initial  value,  namely  PH  5.  Similar  results  were 
obtained  by  Hoagland4  with  barley  plants.  Later  work  shows  that 
solutions  of  (NH4)  CI,  K.,S04  and  some  other  salts  behave  similarly 
to  (NH4)2S04.  The  reaction  may  even  change  appreciably  toward  a 
higher  acidity  especially  when  the  plants  have  not  been  previously 
grown  in  a  complete  culture  solution.  When  complete  culture  solu- 
tions were  used,  the  reaction  was  invariably  changed  toward  neutral- 


438  University  of  California  Publications  in  Agricultural  Sciences       [Vol.4 

ity.  These  results  with  complete  culture  solutions  were  confirmed  by 
Duggar2  and  several  later  workers  for  different  types  of  plants  and 
solutions.  Jones  and  Shive5  found  that  the  reactions  of  20  repre- 
sentative solutions  of  the  Tottingham  series  were  changed  toward 
neutrality.  When,  however,  (NH4)2S04  was  substituted  for  KNO. 
in  these  solutions,  the  reactions  remained  practically  constant  at  the 
original  value,  namely  PH  4.8. 

In  the  present  investigation,  all  the  plants  experimented  with 
invariably  changed  the  reaction  of  the  complete  culture  solution 
toward  some  point  between  PH  6.5  and  PH  6.9,  irrespective  of  what 
the  original  concentration  might  have  been   (see  Part  I). 

The  exact  mechanism  by  which  the  plant  changes  the  reaction  of 
a  solution  has  not  been  established.  In  a  uni-salt  solution,  this  may 
he  ascribed  to  ionic  exchanges,  and  on  the  alkaline  side,  the  OH  ions 
are  partly  neutralized  by  carbon  dioxide  excreted  from  the  roots. 
In  a  complete  culture  solution,  the  problem  becomes  more  complex.  It 
will  thus  be  of  advantage  to  tabulate  the  different  methods  which  a 
growing  plant  conceivably  might  have  at  its  disposal  for  changing 
the  reaction  of  the  solution. 

The  decrease  of  H  ion  concentration  would  be  accomplished: 

1.  By  neutralizing  II  ions  by  OH  ions  derived  from  some  base 
excreted  by  the  roots  or  from  dead  root  cells. 

2.  By  absorbing  H  ions  and  simultaneously  replacing  these  by 
some  other  positive  ion. 

3.  By  absorbing  an  anion  and  excreting  OH  ions  simultaneously. 

4.  By  absorbing  H  ions  and  an  equivalent  amount  of  some  negative 
ion. 

5.  By  absorbing  an  anion  and  excreting  simultaneously  another 
anion  which  forms  an  acid  with  a  lower  degree  of  dissociation  or  an 
acid  which  is  volatile  under  the  conditions. 

The  H  ion  concentration  is  increased: 

(a)  If  OH  ions  are  neutralized  by  II  ions  derived  from  some  acid 
excreted  by  the  plant  or  from  dead  root  cells. 

(6)  If  OH  ions  are  absorbed  but  simultaneously  replaced  by  some 
other  anion. 

(c)  If  OH  ions  and  an  equivalent  amount  of  a  cation  are  absorbed 
simultaneously. 

(<7)    If  a  positive  ion  is  absorbed  and  replaced  by  H  ions. 

Excretions  by  the  roots  are  confined  to  the  acid  HOO.r  (or  C02) 
and,    under    certain    conditions,    small    amount    of    cations,    notably 


1924]     Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        43(» 

calcium.  The  quantities  of  the  latter  are,  however,  insufficient  to 
account  for  more  than  a  very  small  part  of  the  power  of  the  plant  to 
change  the  reaction  of  the  solution.  The  increase  in  the  PH  value 
must  thus  be  accounted  for  by  methods  3,  4,  or  5. 

The  decrease  in  the  Pn  value  of  a  culture  solution  might  take  place 
by  any  or  all  of  the  methods  outlined  under  a,  b,  c,  and  d.  In  some 
cases  the  PH  value  of  the  solution  is  decreased  to  aboiit  PH  3.2,  as 
frequently  happens  in  a  uni-salt  solution  of  K2S04,  for  example.  Since 
the  concentration  of  the  OH  ions  is  very  small  at  this  reaction,  it  is 
possible  that  method  <1  is  chiefly  involved.  Method  c,  however,  can- 
not be  excluded  from  consideration. 

The  plant  has  a  very  efficient  means  at  its  disposal  for  reducing 
the  alkalinity  of  a  solution  in  that  it  normally  excretes  relatively  large 
amounts  of  C02  (method  a).  This,  however,  is  not  the  only  mechanism 
involved  as  is  apparent  from  the  results  of  the  following  experiment. 
Corn  plants  growing  in  a  complete  culture  solution  maintained  at 
PH  8.5,  as  was  described  in  Part  I,  neutralized  within  one  week  0.0257 
equivalents  of  alkali.  When  the  solution  was  analyzed  only  0.0185 
equivalents  of  C02  were  found.  Hence  approximately  one-fifth  of 
the  alkali  added  must  have  been  neutralized  by  methods  c  and  <L 
Whether  this  neutralization  was  brought  about  with  either  or  both  of 
these  methods,  the  final  composition  of  the  solution  would  be  the 
same.  The  solution  must  have  lost  approximately  0.007  equivalents 
of  cations,  except  H-ion  and  an  equivalent  amount  of  OH  ions. 

The  conclusion,  then,  is  inevitable  that,  exclusive  of  the  H  and  OH 
ions,  greater  equivalent  proportions  of  anions  than  of  cations  must 
be  absorbed  on  the  acid  side.  On  the  alkaline  side,  the  reverse  must 
be  true. 

The  rate  of  absorption  of  either  the  anions  or  the  cations,  or  both, 
may  be  influenced  by  differences  in  the  reaction  of  the  culture  medium 
in  order  to  bring  about  this  selective  absorption. 

Several  experiments  Avere  carried  out  to  obtain  some  preliminary 
information  on  this  point.  The  problem  was  attacked  by  means  of 
absorption  studies,  the  relative  amounts  of  the  different  ions  absorbed 
at  different  reactions  by  similar  plants  being  determined.  For  the 
purpose  of  these  experiments,  actively  growing  four-week-old  plants 
were  used.  The  plants  were  grown  in  earthenware  crocks,  the  solu- 
tion used  being  identical  with  the  culture  solution  described  in  Part  T, 
except  that  KNO..  was  substituted  for  NaN03.  The  reactions  were 
maintained   at    certain    definite   values   by    means    of   the    technique 


44(1 


University  of  California  Publications  in  Agricultural  Sciences       [Vol.4 


described  in  Part  I,  and  the  absorption  was  allowed  to  take  place 
over  a  period  of  from  3  to  4  days,  after  which  the  solutions  were 
made  up  to  the  original  volume  and  analyzed. 

The  results  of  experiments  with  barley  and  cucumber  plants  are 
given  in  tables  1  and  2,  respectively.  Similar  experiments  were  carried 
out  with  peas. 

TABLE   1 
Absorption  of  Anions  and  Cations  by  Barley  Plants  at  Different  Reactions 


No.  of 

plants 

Pn  at  which 
solution  was 
maintained 

Total  weights  absorbed,  gms. 

Period  of 
absorption 

NOs 

PO( 

K 

Ca 

Mg 

3  days 
3  days 

30 
30 

4.5 

8.0 

.7144 
.5952 

.1824 
.1856 

.222 
.293 

-.018 
.006 

.023 
.038 

TABLE  2 

Absorption  of  Anions  and  Cations  by  Cucumber  Plants  at 

Different  Reactions 


No.  of 
plants 

Ph  at  which 
solution  was 
maintained 

Total  weights  absorbed,  gms. 

Period  of 
absorption 

NOs 

PO( 

K 

Ca 

3  days 
3  days 
3  days 

25 
25 
25 

5.0 
6.0 
7.0 

.2557 
.2425 
2020 

.0646 
.0420 
.0720 

.060 
.105 
.180 

-.0010 
.0101 
.0169 

It  is  apparent  that  the  influence  of  the  reaction  is  most  marked 
on  the  rate  of  the  absorption  of  the  cations.  Invariably  there  were 
more  cations  absorbed  from  the  alkaline  than  from  the  acid  solutions. 
i.e..  the  rate  of  absorption  of  cations  was  increased  by  a  decrease  in 
the  concentration  or  activity  of  the  H  ion  and  vice  versa.  This  implies 
a  relative  increase  of  the  activity  of  the  cations  in  the  solutions  over 
that  in  the  plant.  It  is  more  probable  that  the  activity  of  the  cations 
in  the  plant  is  decreased  than  that  the  activity  of  the  cations  in  the 
solution  is  increased  so  as  to  bring  about  such  a  marked  change  in 
the  rate  of  absorption  of  the  cations. 

Loeb's"  brilliant  researches  have  thrown  much  light  on  the  rela- 
tion existing  between  inorganic  salts  or  ions  and  charged  organic 
colloids  and  the  distribution  of  ions  on  the  two  sides  of  a  membrane 
when  one  side  contains  an  ion  which  cannot  diffuse  through  the  mem- 
brane. To  what  extent  the  principles  discovered  by  him  may  apply 
to  the  absorption  of  salts  by  plants  it  is  impossible  to  say  at  present. 
Tf  it  he  assumed  that  ionic  equilibria  are  established  between  the  roots 


1824]     Theron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        441 

and  the  solution,  these  principles  will  undoubtedly  determine  the 
equilibrium  concentration  of  the  ions.  Unfortunately,  however,  the 
existence  of  such  equilibria  in  plant  cells  has  not  been  established 
definitely.  Assuming,  however,  that  an  equilibrium  is  established 
between  the  ions  in  the  cells,  which  are  active  in  absorption,  and  the 
ions  in  the  solution,  the  effect  of  the  reaction  on  the  rate  of  absorption 
of  the  cations  is  readily  explained  as  will  be  apparent  from  the 
following  considerations.* 

In  the  cell  there  are,  among  other  substances,  anions  of  organic 
acids,  salts  of  these  acids  and  probably  of  free  acids,  to  which  the 
membranes  effective  in  the  absorption  are  impermeable,  and  also  com- 
plex colloidal  bodies  which  are  probably  negatively  charged  (see 
below).  Since  the  reaction  of  the  cells  of  the  roots  (in  so  far  as  it  is 
reflected  in  the  plant  juices)  is  influenced  markedly  by  the  reaction  of 
the  solution  (see  Part  I),  it  is  clear  that  an  acid  reaction  of  the  culture 
solution  will  have  the  effect  of  depressing  the  dissociation  of  these 
acids  and  negatively  charged  bodies,  and  consequently  the  number 
of  cations  held  by  electrostatic  forces  will  be  decreased,  i.e.,  the 
activity  of  the  cations  will  be  increased.  In  other  words,  the  rate 
of  absorption  of  the  cations  will  be  decreased  by  an  increase  in  the 
H  ion  concentration.  As  a  rule,  a  greater  number  of  equivalents  of 
nitrate  ions  were  absorbed  on  the  acid  than  on  the  alkaline  side, 
whereas  the  absorption  of  phosphate  ions  was  very  irregular.  Unfor- 
tunately, the  ease  with  which  a  plant  is  able  to  replace  certain  anions 
absorbed  by  HC03  ions  complicates  attempts  to  determine  whether  or 
not  the  rate  of  absorption  of  anions  is  affected  by  the  reaction  of  the 
culture  solution.  If  the  root  contains  any  positively  charged  bodies 
to  which  the  membranes  are  not  permeable,  we  would  expect  an 
increase  in  the  H  ion  concentration  to  produce  an  increased  rate  of 
absorption  of  the  anions. 

The  charge  on  the  proteins  and  other  amphoteric  bodies  which 
constitute  protoplasm  becomes  of  paramotuit  importance  in  this  con- 
nection. To  gain  some  information  on  this  point  resort  was  made  to 
cataphoresis  experiments.  A  slightly  modified  form  of  the  apparatus 
described  by  Cohn,  Gross,  and  Johnson1  was  used  for  the  purpose, 


*  Since  this  paper  was  completed,  work  has  been  reported  by  the  Laboratory 
of  Plant  Physiology  of  Harvard  University  and  by  the  Laboratory  of  Plant 
Nutrition  of  the  University  of  California,  which  indicates  that  additional 
considerations  must  be  taken  into  account.  For  example,  experiments  on  the 
alga  Nitella  (from  which  uncontaminated  cell  sap  may  be  obtained)  prove 
that  an  ion  may  be  absorbed  from  a  solution  of  low  concentration  into  a 
solution  of  high  concentration,  and  that  certain  inorganic  elements,  such  as 
potassium,  exist  in  the  cell  almost  entirely  in  ionic  form. 


44ii  University  of  California  Publications  in  Agricultural  Sennas       [Vol.  4. 

and  the  migration  of  the  nitrogenous  constituents  in  the  sap,  expressed 
from  the  roots  after  freezing,  was  determined  by  analyzing  the  buffer 
mixtures  in  the  cathodic  and  anodic  chambers  for  nitrogen  by  the 
Kjeldahl  method.  In  the  case  of  the  juices  from  the  roots  of  barley, 
pea,  and  cucumber  plants,  the  migration  was  invariably  found  to  be 
toward  the  anode,  proving  that  these  bodies  are  charged  negatively 
at  the  reaction  at  which  they  occur  in  the  plant.  The  direction  of 
migration  was  not  reversed  at  a  reaction  of  PH  4.5.  The  same  results 
were  found  when  the  root  juices  were  well  dialysed  against  distilled 
water.  Since  the  reaction  of  the  juice,  when  it  is  freshly  expressed 
from  the  tissue,  has  a  reaction  of  approximately  PH  6,  it  is  plain  that 
the  isoelectric  points  of  the  nitrogenous  bodies  are  considerably  below 
the  Ph  values  at  which  they  normally  occur  in  the  sap. 

Deductions  drawn  from  experiments  with  the  expressed  sap  can 
hardly  be  considered  as  applying  to  the  living  root,  which  is  a  highly 
differentiated  structure.  The  process  of  freezing  may  bring  about 
changes  sufficiently  severe  to  change  the  sign  of  the  charge  on  some 
of  the  ampholytes  in  the  living  cell  or  to  cause  mutual  precipitation 
of  oppositely  charged  colloids  from  the  same  or  from  different  cells. 
In  general  it  is  improbable,  however,  that  the  isoelectric  points  of  the 
different  ampholytes  will  be  changed  materially  by  this  treatment. 
Since  the  former  are  so  far  removed  from  the  reaction  at  which  these 
ampholytes  occur  in  the  root  tissues,  it  is  highly  probable  that  the 
majority  of  the  proteins  and  other  nitrogenous  bodies  are  charged 
negatively  in  the  living  cell,  and  that  the  sign  of  the  charge  is  not 
readily  reversible  as  assumed  by  Haynes.3  The  work  of  Meier7  sub- 
stantiates the  above  conclusions.  This  investigator  found  that  the 
cell  contents  in  the  roots  of  actively  growing  plants  moved  under  the 
influence  of  a  small  current  as  if  they  were  negatively  charged. 

If  a  plant  be  allowed  to  change  the  reaction  of  an  acid  or  alkaline 
solution,  a  certain  PH  must  be  reached  at  which  the  tendency  of 
methods  3,  4,  and  5  to  decrease  the  II  ion  concentration  is  balanced 
by  the  tendency  of  methods  a,  c,  and  <1  to  decrease  the  OH  ion  con- 
centration. Because  of  the  many  factors  involved  in  this  equilibrium, 
one  can  hardly  expect  this  reaction  to  he  very  definite  under  the 
varying  conditions  of  experimentation.  For  barley  and  corn,  this 
value  was  found  to  be  PH  6.75  to  PH  6.8,  and  for  peas,  PH  6.65  to 
Ph  6.7. 

The  significance  of  this  point  is  not  known  at  present.  The  main 
factor  involved  in  bringing  about  this  reaction  in  a  solution  seems  to 


J 924]      Tlieron:  Inter-relations  Between  the  Plant  and  Its  Culture  Medium        443 

be  the  activities  of  the  C02,  H,CO.„  and  the  ions  of  this  acid  in  the 
plant  and  in  the  solution.  If  this  equilibrium  is  disturbed  in  such 
a  way  as  to  allow  the  escape  of  CO,,  as  may  happen  when  the  volume 
of  the  solution  is  diminished  excessively  by  transpiration,  the  PH 
value  must  rise.  This  is  easily  demonstrated  by  allowing:  the  solutions 
in  the  containers  to  '  run  down. '  The  reaction  may  rise  to  as  high  as 
PH  8.5.  On  the  other  hand,  if  the  other  factors  which  contribute 
toward  this  equilibrium  be  missing,  the  PH  value  will  decrease  till  an 
equilibrium  is  established  between  the  CO,  of  the  atmosphere  above 
the  solution  and  the  H,C03  and  HCO  r  in  the  solution.  Such  a  con- 
dition is  brought  about  in  distilled  water  in  which  the  reaction  is 
maintained  at  a  slight  acidity.  The  equilibrium  reaction  also  depends 
upon  the  rapidity  with  which  the  different  ions  are  absorbed.  In 
solutions  in  which  the  rapidly  absorbed  anion  N03"  is  replaced  by  the 
rapidly  absorbed  cation  NH4\  as  in  the  investigation  by  Jones  and 
Shive,"'  the  equilibrium  will  naturally  be  thrown  over  to  the  acid  side. 

From  the  above  considerations,  it  is  obvious  that  an  'optimum' 
culture  solution  for  the  growth  of  plants  will  depend  not  only  on  the 
composition  of  the  culture  solution,  but  also  upon  the  partial  pressure 
of  the  CO,  in  the  atmosphere  and  other  atmospheric  conditions. 

The  fact  that  the  optimum  reaction  for  the  growth  of  plants  in 
solution  culture  is  on  the  acid  side  is  possibly  correlated  in  part  with 
the  greater  ease  with  which  the  respiratory  C02  can  diffuse  out  and 
away  from  the  roots  at  this  reaction.  If  this  theory  is  correct,  the 
optimum  reaction  will  even  be  slightly  more  toward  the  acid  side  in 
soil,  since  the  diffusion  of  CO,  is  interfered  with. 


SUMMARY 

1.  A  study  of  the  effect  of  the  reaction  of  the  solution  on  the 
absorption  of  the  anions  and  cations  by  the  plant  is  described. 

2.  Several  methods  are  outlined  by  which  the  plant  changes  the 
reaction  of  either  acid  or  alkaline  culture  solutions  toward  neutrality. 

3.  Absorption  experiments  show  that  the  rate  of  absorption  of 
the  cations  is  increased  by  a  decrease  in  the  H-ion  concentration, 
while  the  ability  of  the  plant  to  excrete  CO,  from  the  roots  allows  of 
the  selective  absorption  of  anions  from  the  acid  solutions. 

4.  The  charge  on  the  constituents  of  the  root  cells  may  be  assumed 
to  be  of  vital  importance  in  the  mechanism  of  absorption. 


444  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

5  The  nitrogenous  constituents  of  the  cell  sap  are  charged  nega- 
tively and  the  isoelectric  points  of  the  majority  of  ampholytes  in  the 
cell  is  below  PH  4.5. 

6  Pea  plants  change  the  reaction  of  either  acid  or  alkaline  solu- 
tions from  PH  6.65  to  PH  6.7,  whereas  barley  and  corn  plants  change 
it  from  PH  6  75  to  PH  6.8.  The  main  factor  involved  in  bringing 
about  this  reaction  is  the  CO.-HCO,-  equilibrium  in  the  plant  and  in 

the  solution. 

Transmitted,  March  S3,  1923. 


LITERATURE  CITED 

i  Cohn   E.  J.,  Gross,  J.,  and  Johnson,  O.  C.  _   . 

1019.     The  isoelectric  points  of  the  proteins   in  certain  vegetable   juices. 
Jour.  Gen.  Physiol.,  vol.  2,  no.  2,  pp.  145-160. 

^ToS'VL  concentration  and  the  composition  of  nutrient  solutions  in 
relation  to  the  growth  of  seed  plants.  Ann.  Musonn  Bot. 
Garden,  vol.  7,  no.  1,  pp.  1-49. 

3H™'  ^he  action  of  salts  and  non-electrolytes  upon  buffer  solutions  and 
amphoteric  electrolytes  and  the  relation  of  these  effects  to  the 
permeability  of  the  cell.  Biochem.  Jour.,  vol.  15,  no.  3,  pp. 
440-461. 

4H01919NDEeiafion  of  the  concentration  and  reaction  of  the  nutrient  medium 
to  the  growth  and  absorption  of  the  plant.  Jour.  Agr.  Res, 
vol.  18,  pp.  73-117. 

'  JT921L-  STLfSil  sulfate  Upon  plants  in  nutrient  solutions  sup^ 
plied  with  ferrie  phosphate  and  ferrous  sulfate  as  sources  ot 
iron.    Jour.  Agr.  Res,  vol.  21,  no.  10,  pp.  701-728. 

6LO?918:  Amphoteric  colloids.  Jour.  Gen.  Physiology,  vol.  1,  pp.  39-60  237- 
254,  363-385,  559-580;  and  subsequent  papers  dealing  with  tbe 
same  general  subject. 

™ESE21HEFffett'  of  direct  current  on  cells  of  root  tips  of  Canada  field  pea. 
Bot.  Gaz,  vol.  72,  no.  3,  pp.  113-138. 

8PA19irUebt  lonen  Aufnahme.     Jahrl,  Wiss.  Bot.   (Pringsheim) ,  Band  56, 

pp.  689-733. 


